A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor

Abstract

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the ␣5 helix of G␣ i1 , especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in G␣ i1 by inserting 4 alanine amino acids into the ␣5 helix between residues Gln 333 and Phe 334 (Ins4A). This extends the length of the ␣5 helix without disturbing the ␤6-␣5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His 57 , Phe 189 , Phe 191 , and Phe 336 ; it also disturbs the rotation of the ␣5 helix and the -interaction between His 57 and Phe 189 . In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between ␣5, ␣1, and ␤2-␤3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the ␣5 helix for G protein release from the receptor after GTP binding.Heterotrimeric G proteins, composed of ␣, ␤, and ␥ subunits, act as a molecular switches that turn on intracellular signaling cascades in response to the activation of G protein-coupled receptors by extracellular stimuli. Therefore, G proteins have a critical role in many different cellular responses (1-6).The G␣ subunit binds GDP and forms a tight complex with the G␤␥ subunits. Activated G protein-coupled receptors can catalyze the exchange of GDP for GTP, which leads to the dissociation of the receptor-G protein complex into isolated receptor and G␣ and G␤␥ subunits. Both the G␣ and G␤␥ subunits can then stimulate or inhibit downstream effectors. Signal propagation ceases after the G␣ subunit hydrolyzes GTP, returns to the inactive state, and rebinds to the G␤␥ subunit, regenerating the GDP-bound heterotrimeric state.Previous studies showed that the activated receptor directly interacts with the G protein by binding to the C-terminal ␣5 helix of G␣, inducing a rigid body rotation and translation that pull this helix into a hydrophobic pocket on the receptor (7,8). This leads to the rearrangement of the interfaces between helices ␣5, ␣1, and the ␤2-␤3 strands and between ␣5 and the ␤6-␣5 loop (1, 7, 9 -11). Residue Phe 336 in the ␣5 helix is highly conserved in small (12, 13) and large GTPases (14) in both the animal and plant kingdoms (15-18). Our in silico results predicted that Phe 336 is the most energetically important residue both in maintaining the basal state and in promoting the receptor-bound conformation (6...

Structural basis for GPCR-independent activation of heterotrimeric Gi proteins

“…The structural basis for GPCR-dependent G protein activation had challenged the field for decades but was revealed in the past 8 y by a series of landmark structural studies (14)(15)(16)(17)(18). These studies have demonstrated that one of the key mechanisms of G protein activation by GPCRs involves perturbation of the so-called hydrophobic core (19,20) of the Gα GTPase domain, which is mediated by displacement of the Cterminal α5 helix and insertion of the GPCR's intracellular loop 2 (14)(15)(16)(17)(18).…”

“…The most striking increase in dynamics was observed in the C-terminal region of Sw-I and the β2-β3 strands ( Fig. 6 B and C), which normally pack against the α1and α5-helices of Gαi to form the hydrophobic core (20) of the GTPase domain. When simulations were run in the presence of the His-tag linker, the dynamics of Sw-II was unchanged with respect to the GIV-bound state, the high mobility of Sw-III was restored to the GDP-only level, and the GIV-induced increase in Sw-I and β2-β3 strand dynamics was partially negated, in agreement with a potential role of the His-tag linker in stabilizing GIV-GEM-bound Gαi and facilitating its crystallization (SI Appendix, Fig.…”

Latrophilins are essential for endothelial junctional fluid shear stress mechanotransduction

“…proteins dissociate from GPCRs immediately after GTP loading, making affinity purification problematic. We therefore made WT and Q307K Gi1 with the 4 alanine insertion (hereafter ins4A) in helix !5 that increases affinity for GPCRs even in the presence of GTP 19 . These G!…”

Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy

“…By validation of calculated infrared (IR) spectra from quantum-mechanics/molecular-mechanics (QM/MM) calculations against the experimental values that were measured with natural GTP it will be possible to clarify the structure of the native binding pocket of G α i1 with sub-Å resolution and charge shifts that are important for catalysis can be obtained. Furthermore calculation of the inactive GDP state might clarify the discussion if the inactive state of G α i1 does or does not carry a Mg 2+ ion (PDB: 1GP2 , 5KDO ) ( 9 , 10 , 11 ). There are several examples for the calculation of GTPase IR-spectra in the literature, e.g., for the small GTPases Ras ( 12 , 13 , 14 , 15 , 16 , 17 ), Ran ( 18 ), Arl ( 19 ), and others.…”